Bioprotection using lactobacillus rhamnosus strains

ABSTRACT

The present invention is related to the field of bioprotection, in particular to the strain of  Lactobacillus rhamnosus  CHCC5366 with accession no. DSM23035. Furthermore, the present invention concerns an antifungal composition comprising the strain, an antifungal composition comprising the strain and at least one strain of  Lactobacillus paracasei , food, feed and pharmaceutical products comprising such an antifungal composition, a method of manufacturing such food, feed and pharmaceutical products, a method for reducing the content of yeasts and molds of such food, feed and pharmaceutical products and uses of the antifungal composition.

FIELD OF THE INVENTION

The present invention is related to the field of bioprotection, inparticular to the strain of Lactobacillus rhamnosus CHCC5366 withaccession no. DSM23035. Furthermore, the present invention concerns anantifungal composition comprising the strain, an antifungal compositioncomprising the strain and at least one strain of Lactobacillusparacasei, food, feed and pharmaceutical products comprising such anantifungal composition, a method of manufacturing such food, feed andpharmaceutical products, a method for reducing the content of yeasts andmolds of such food, feed and pharmaceutical products and uses of theantifungal composition.

BACKGROUND OF THE INVENTION

For many years lactic acid bacteria have been used for increasing theshelf life of food products. During fermentation the lactic acidbacteria produce lactic acids and other organic acids thereby reducingthe pH of the food product making it unfavorable to the growth ofunwanted microorganisms, such as pathogenic bacteria, yeasts and fungi.

Additionally, some lactic acid bacteria also produce metabolites withantimicrobial activity.

European Patent Application no. EP0221499 describes the antifungalproperties of Lactobacillus rhamnosus NRRL-B-15972 which is capable ofinhibiting the growth of different molds when cultured on agar mediumsupplemented with cucumber juice.

European Patent Application no. EP0576780 is related to Lactobacillusrhamnosus LC-705 which can inhibit the growth of Penicillium,Cladosporium, Fusarium and Candida on a lactoserum-based medium,supplemented with casein hydrolysate and with yeast extract.

European Patent Application no. EP1442113 is directed to mixtures ofPropionibacterium jensenii and Lactobacillus sp., such as Lactobacillusrhamnosus, with antimicrobial activities for use for bioprotection.

However, there is still a need for bioprotective agents with an improvedantifungal effect as single strains or in combination with otherbioprotective strains.

SUMMARY OF THE INVENTION

The object of the present invention is the provision of novel strains oflactic acid bacteria with a high efficacy as bioprotective agents.

The present inventors have by extensive screening and research foundthat certain Lactobacillus rhamnous strains have a significantly highereffect against yeasts and molds as compared to commercial bioprotectivecultures on the market as well as potassium sorbate, a commonly usedchemical preservative.

The present inventors have further found that a certain group of lacticacid bacteria when combined with another group of lactic acid bacteriaexhibit a significant synergistic antimicrobial effect. Theantimicrobial effect of the two groups of bacteria combined,surprisingly, is greater than the sum of the individual effects of thetwo groups of bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts growth of molds in yoghurt with 1.5% fat fermented with astarter culture YF-L901 alone (top row), together with HOLDBAC™ YM-B(middle row) and together with Lactobacillus rhamnosus CHCC5366. Thetarget contaminants were added in the concentrations mentioned in thetext from left to right: Penicillium brevicompactum (M1), Penicilliumcommune (M6), Acromyrmex versicolor (M7) and Penicillium crustosum(M10), respectively. The yoghurts had been incubated at 7±1° C. for 45days.

FIG. 2 depicts cell counts of a Debaromyces hansenii isolate added toyoghurt with 1.5% fat fermented with a starter culture YF-L901 alone(Reference) or together with the following strains: HOLDBAC™ YM-B(HoldBac YM-B), HOLDBAC™ YM-C(HoldBac YM-C) or Lactobacillus rhamnosusCHCC5366 (CHCC 5366). Yoghurts were stored at 7±1° C. and analyzed atsuitable intervals.

FIG. 3 shows growth of molds on plates prepared from milk fermented witha starter culture alone (reference, first picture), together with Lb.paracasei CHCC14676 (second picture), together with Lb. rhamnosusCHCC5366 (third picture) or together with a combination of Lb. paracaseiCHCC14676 and Lb. rhamnosus CHCC5366 (fourth picture). The targetcontaminants were added in the concentrations mentioned in the text fromtop left and moving clock-wise to the bottom left of the plate:Penicillium nalgiovese, Penicillium commune, Aspergillus versicolor andPenicillium crustosum, respectively. The plates had been incubated at7±1° C. for 12 days.

FIG. 4 shows the growth of: (1) Penicillium solitum, (2) P. palitans,(3) P. discolor, (4) P. spathulatum, (5) P. commune, (6). P. crustosum,(7) P. paneum, (8) P. roqueforti in a reference yoghurt (left column), ayoghurt made with 0.023% potassium sorbate (middle column) and a yoghurtmade with 1E+07 cfu/g of CHCC5366 (right column) after 60 daysincubation at 7° C. when inoculated in concentrations of approx. 1000spores/cup.

FIG. 5 shows growth of Mucor ssp. on Greek yoghurt prepared from milkfermented with a starter culture only (left cup) or from milk fermentedin the presence of Lactobacillus rhamnosus CHCC5366 and Lactobacillusparacasei CHCC14676 (right cup).

FIG. 6 shows growth of the mold Rhizopus stolonifer on Greek yoghurtprepared from milk fermented with a starter culture only (R, i.e.reference), with the starter culture and HOLDBAC™ YM-B (YM-B), with thestarter culture and Lactobacillus rhamnosus CHCC5366 (5366), or with thestarter culture and a combination of Lactobacillus rhamnosus CHCC5366and Lactobacillus paracasei CHCC14676 (5366+14676).

FIG. 7 shows growth of Saccharomyces cerevisiae on white brine cheesemade from milk fermented only with starter culture (reference); withstarter culture and HOLDBAC™ YM-B (YM-B); or with starter culture,Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366(5366+14676).

FIG. 8 shows growth of Kluyveromyces maxianus on white brine cheese madefrom milk fermented only with starter culture (reference); with starterculture and HOLDBAC™ YM-B (YM-B); or with starter culture, Lactobacillusparacasei CHCC14676 and Lactobacillus rhamnosus CHCC5366 (5366+14676).

FIG. 9 shows growth of Penicillium commune on white brine cheese madefrom milk fermented only with starter culture (R, i.e. reference); withstarter culture and HOLDBAC™ YM-B (YM-B); or with starter culture,Lactobacillus paracasei CHCC14676 and Lactobacillus rhamnosus CHCC5366(5366+14676).

FIG. 10 shows growth of P. crustosum on white brine cheese made frommilk fermented only with starter culture (R, i.e. reference); withstarter culture and HOLDBAC™ YM-B (YM-B); or with starter culture andLactobacillus rhamnosus CHCC5366 (5366).

FIG. 11 depicts growth of the mold Penicillium paneum in yoghurt with1.5% fat prepared from milk fermented with a starter culture alone (R,i.e. reference), with the starter culture and HOLDBAC™ YM-B (YM-B), withthe starter culture and Lactobacillus rhamnosus CHCC5366 (5366), or withthe starter culture and a combination of Lactobacillus rhamnosusCHCC5366 and Lactobacillus paracasei CHCC14676 (5366+14676).

FIG. 12 shows growth of the mold Penicillium crustosum in yoghurt with1.5% fat prepared from milk fermented with a starter culture alone (toprow), with starter culture and HOLDBAC™ YM-B (second row), with starterculture and Lactobacillus rhamnosus CHCC5366 (third row) or with starterculture and a combination of Lactobacillus rhamnosus CHCC5366 andLactobacillus paracasei CHCC14676 (bottom row). The target contaminantwas added in the concentrations of 100 spores/cup and the yoghurts hadbeen incubated at 7±1° C. (left column), 12±1° C. (centre column) or22±1° C. (right column) for 36 days.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid. The industrially most useful lactic acidbacteria are found within the order “Lactobacillales” which includesLactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostocspp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp. andEnterococcus spp. These are frequently used as food cultures alone or incombination with other lactic acid bacteria.

The term “food” is meant to include also cheese. The term “cheese” isunderstood to encompass any cheese, including hard, semi-hard and softcheeses, such as cheeses of the following types: Cottage, Feta, Cheddar,Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, bluecheeses, brine cheeses, Camembert and Brie. The person skilled in theart knows how to convert the coagulum into cheese, methods can be foundin the literature, see e.g. Kosikowski, F. V., and V. V. Mistry, “Cheeseand Fermented Milk Foods”, 1997, 3rd Ed. F. V. Kosikowski, L. L. C.Westport, Conn. As used herein, a cheese which has a NaCl concentrationbelow 1.7% (w/w) is referred to as a “low-salt cheese”.

Lactic acid bacteria, including bacteria of the species Lactobacillussp. and Lactococcus sp., are normally supplied to the dairy industryeither as frozen or freeze-dried cultures for bulk starter propagationor as so-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of adairy product, such as a fermented milk product or a cheese. Such lacticacid bacterial cultures are in general referred to as “starter cultures”or “starters”.

The term “mesophile” herein refers to microorganisms that thrive best atmoderate temperatures (15° C.-40° C.). The industrially most usefulmesophilic bacteria include Lactococcus spp. and Leuconostoc spp. Theterm “mesophilic fermentation” herein refers to fermentation at atemperature between about 22° C. and about 35° C. The term “mesophilicfermented milk product” refers to fermented milk products prepared bymesophilic fermentation of a mesophilic starter culture and include suchfermented milk products as buttermilk, sour milk, cultured milk,smetana, sour cream and fresh cheese, such as quark, tvarog and creamcheese.

The term “thermophile” herein refers to microorganisms that thrive bestat temperatures above 43° C. The industrially most useful thermophilicbacteria include Streptococcus spp. and Lactobacillus spp. The term“thermophilic fermentation” herein refers to fermentation at atemperature above about 35° C., such as between about 35° C. to about45° C. The term “thermophilic fermented milk product” refers tofermented milk products prepared by thermophilic fermentation of athermophilic starter culture and include such fermented milk products asset-yoghurt, stirred-yoghurt and drinking yoghurt.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as cows, sheep, goats, buffaloes or camels. Ina preferred embodiment, the milk is cow's milk. The term milk alsoincludes protein/fat solutions made of plant materials, e.g. soy milk.

The term “milk substrate” may be any raw and/or processed milk materialthat can be subjected to fermentation according to the method of theinvention. Thus, useful milk substrates include, but are not limited to,solutions/suspensions of any milk or milk like products comprisingprotein, such as whole or low fat milk, skim milk, buttermilk,reconstituted milk powder, condensed milk, dried milk, whey, wheypermeate, lactose, mother liquid from crystallization of lactose, wheyprotein concentrate, or cream. Obviously, the milk substrate mayoriginate from any mammal, e.g. being substantially pure mammalian milk,or reconstituted milk powder.

Prior to fermentation, the milk substrate may be homogenized andpasteurized according to methods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. If homogenization is performed prior tofermentation, it may be performed so as to break up the milk fat intosmaller sizes so that it no longer separates from the milk. This may beaccomplished by forcing the milk at high pressure through smallorifices.

“Pasteurizing” as used herein means treatment of the milk substrate toreduce or eliminate the presence of live organisms, such asmicroorganisms. Preferably, pasteurization is attained by maintaining aspecified temperature for a specified period of time. The specifiedtemperature is usually attained by heating. The temperature and durationmay be selected in order to kill or inactivate certain bacteria, such asharmful bacteria. A rapid cooling step may follow.

“Fermentation” in the methods of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the methods of theinvention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of dairy products arewell known and the person of skill in the art will know how to selectsuitable process conditions, such as temperature, oxygen, amount andcharacteristics of microorganism(s) and process time. Obviously,fermentation conditions are selected so as to support the achievement ofthe present invention, i.e. to obtain a dairy product in solid (such asa cheese) or liquid form (such as a fermented milk product).

The compositions of the invention provide the advantage that unwantedmicroorganisms selected from fungi, bacteria and mixtures thereof, forexample on food, feed and pharmaceutical products and in humans andanimals, can be inhibited. The prevention and/or inhibition of thegrowth of fungi, such as yeasts and molds, is particularly envisaged.Therefore, in a preferred embodiment, the term “antimicrobial” is to beunderstood as “antifungal”.

The term “unwanted microorganisms” herein refers to microorganisms suchas bacteria and fungi, such as yeasts, which are pathogenic and/or ableto deteriorate food, feed or pharmaceutical products. The compositionsof the invention provide the advantage that unwanted microorganismsselected from fungi, bacteria and mixtures thereof, for example on food,feed and pharmaceutical products and in humans and animals, can beinhibited. The prevention and/or inhibition of the growth of fungi, suchas yeasts and molds, is particularly envisaged.

The terms “to inhibit” and “to be inhibiting” in relation to yeasts andmolds mean for example that the growth or the number or theconcentration of yeasts and molds, for example in food products and/oron the surface of food products comprising the strains according to thepresent invention, is lower than in food products and/or on the surfaceof food products which do not comprise such strains.

In the present context, the term “mutant” should be understood as astrain derived from a strain of the invention by means of e.g. geneticengineering, radiation and/or chemical treatment. It is preferred thatthe mutant is a functionally equivalent mutant, e.g. a mutant that hassubstantially the same, or improved, properties (e.g. regardingantifungal properties) as the mother strain. Such a mutant is a part ofthe present invention. Especially, the term “mutant” refers to a strainobtained by subjecting a strain of the invention to any conventionallyused mutagenization treatment including treatment with a chemicalmutagen such as ethane methane sulphonate (EMS) orN-methyl-N′-nitro-N-nitroguanidine (NTG), UV light or to a spontaneouslyoccurring mutant. A mutant may have been subjected to severalmutagenization treatments (a single treatment should be understood onemutagenization step followed by a screening/selection step), but it ispresently preferred that no more than 20, or no more than 10, or no morethan 5, treatments (or screening/selection steps) are carried out. In apresently preferred mutant, less that 5%, or less than 1% or even lessthan 0.1% of the nucleotides in the bacterial genome have been shiftedwith another nucleotide, or deleted, compared to the mother strain.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Implementation and Aspects of the Invention

The present inventors have screened among 200 candidates ofLactobacillus plantarum, Lactobacillus paracasei and Lactobacillusrhamnosus to find the most efficient strains against a wide range ofmicroorganisms, such as yeasts and molds.

Screenings were carried out in a model assay mimicking mesophilicfermented milk products as much as possible in milk-based media to whicha relevant starter culture was added with or without bioprotectivecandidates and which was fermented under conditions relevant tomesophilic fermented milk products. Target organisms were isolated frommesophilic fermented milk products. Both the purified lactic acidbacteria from the HOLDBAC™ cultures from Danisco A/S, Denmark, as wellas the complete HOLDBAC™ YM-B and HOLDBAC™ YM-C cultures containing boththe lactic acid bacteria and propionic acid bacteria were used as benchmark.

The model assay is described in European Patent Application no.EP11161609.0.

Seventeen candidates among Lactobacillus paracasei and Lactobacillusrhamnosus were found generally to inhibit the 12 indicator fungi as wellor better than the bench mark lactic acid bacteria when tested at 25° C.

When tested on yoghurt one strain of Lactobacillus rhamnosus,Lactobacillus rhamnosus strain CHCC5366 that was deposited with theGerman Collection of Microorganisms and Cell Cultures (DSMZ) underaccession no. DSM23035 showed to be significantly better at inhibitingyeasts and molds than a commercially available bioprotective culture.

The inhibiting effect of the strain against the yeasts and molds couldbe determined by storing the fermented milk products at a suitabletemperature during a suitable storage time as described in the Examplesbelow.

In general, the suitable temperature at which this method should beperformed depends on the temperature at which the specific food, feed orpharmaceutical product is normally stored and/or manufactured. Thetemperature at which the products are usually stored are between 5° C.and 26° C., preferably the temperature is about 8° C.

The storage time at the temperature depends on the time during which thefood, feed, or pharmaceutical product is normally stored (shelf life).The storage time usually is 5-65, preferably 7-60 days, more preferably7-28 days, and even more preferably the storage time is about 21 days.

Accordingly, a first aspect of the present invention relates to aLactobacillus rhamnosus strain selected from the group consisting of theLactobacillus rhamnosus strain CHCC5366 that was deposited with theGerman Collection of Microorganisms and Cell Cultures (DSMZ) underaccession No. DSM23035 and mutant strains derived thereof.

Thus, apart from the deposited strain mentioned above, the inventionalso pertains to mutants that have been derived from this strain, i.e.they have been obtained by using the deposited strain CHCC5366 as astarting material. The mutant strain may be derived from the depositedstrain, e.g., by means of genetic engineering, radiation, UV light,chemical treatment and/or methods that induce changes in the genome. Amutant according to the invention will inhibit and/or prevent the growthof certain bacteria or fungi, preferably molds. It is preferred that themutant has essentially at least 80% or more, at least 90% or more, atleast 95% or more, or even up to 100% or more of the antifungal effectcompared with its mother strain when determined, e.g., in an assay asdescribed in Example 1 using one of the molds P. brevicompactum, P.commune, A. versicolor, D. hansenii or P. crustosum as a referenceorganism the growth of which is to be inhibited.

It is clear for the skilled person that by using the deposited strain asstarting material, the skilled reader can by conventional mutagenesis orre-isolation techniques routinely obtain further mutants or derivativesthereof that retain the herein described relevant features andadvantages. Accordingly, the term “mutant strains derived thereof” ofthe first aspect relates to mutant strains obtained by using thedeposited strain as starting material.

A second aspect relates to an antifungal composition comprising at leastone Lactobacillus rhamnosus strain according to the first aspect of theinvention, preferably Lactobacillus rhamnosus strain CHCC5366.

When testing Lactobacillus rhamnosus strain CHCC5366 in combination withdifferent Lactobacillus paracasei strains, it was unexpectedly foundthat these combinations were even better than either of the strainsalone even when the total concentrations of cells were identical. In oneembodiment, the invention relates to a combination of Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC12777. In anotherembodiment, the invention relates to a combination of Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676. Thesecombinations seemed to be more efficient than the bench mark culturesHOLDBAC™ YM-B and HOLDBAC™ YM-C from Danisco, Denmark.

Accordingly, in a preferred embodiment the present invention relates toantimicrobial and more preferably antifungal compositions comprisingLactobacillus rhamnosus strain CHCC5366 that was deposited with theGerman Collection of Microorganisms and Cell Cultures (DSMZ) underaccession No. DSM23035 or a mutant derived thereof and at least oneLactobacillus paracasei strain. Preferably, the at least oneLactobacillus paracasei strain is selected from the group consisting ofLactobacillus paracasei strain CHCC14676 with accession no. DSM25612,Lactobacillus paracasei strain CHCC12777 with accession no. DSM24651 andmutant strains derived from these deposited strains.

In one aspect the invention thus provides an antimicrobial and morepreferably antifungal composition comprising Lactobacillus rhamnosusstrain CHCC5366 or a mutant derived thereof and Lactobacillus paracaseistrain CHCC12777 or a mutant derived thereof. In another aspect, theinvention provides an antimicrobial and more preferably antifungalcomposition comprising at least Lactobacillus rhamnosus strain CHCC5366or a mutant derived thereof and Lactobacillus paracasei strain CHCC14676or a mutant derived thereof. Preferably, the combination of theLactobacillus paracasei strain and the Lactobacillus rhamnosus strain inthe compositions of the invention act synergistically in terms of theirantimicrobial and/or antifungal activity.

The antifungal composition typically comprises the bacteria in aconcentrated form including frozen, dried or freeze-dried concentratestypically having a concentration of viable cells, which is in the rangeof 10⁴ to 10¹² cfu (colony forming units) per gram of the compositionincluding at least 10⁴ cfu per gram of the composition, such as at least10⁵ cfu/g, e. g. at least 10⁶ cfu/g, such as at least 10² cfu/g, e.g. atleast 10⁸ cfu/g, such as at least 10⁹ cfu/g, e.g. at least 10¹⁰ cfu/g,such as at least 10¹¹ cfu/g. Thus, the composition of the invention ispreferably present in a frozen, dried or freeze-dried form, e.g. as aDirect Vat Set (DVS) culture. However, as used herein the compositionmay also be a liquid that is obtained after suspension of the frozen,dried or freeze-dried cell concentrates in a liquid medium such as wateror PBS buffer. Where the composition of the invention is a suspension,the concentration of viable cells is in the range of 10⁴ to 10¹² cfu(colony forming units) per ml of the composition including at least 10⁴cfu per ml of the composition, such as at least 10⁵ cfu/ml, e.g. atleast 10⁶ cfu/ml, such as at least 10² cfu/ml, e.g. at least 10⁸ cfu/ml,such as at least 10⁹ cfu/ml, e.g. at least 10¹⁰ cfu/ml, such as at least10¹¹ cfu/ml.

The composition may as further components contain cryoprotectants and/orconventional additives including nutrients such as yeast extracts,sugars and vitamins, e.g. vitamin A, C, D, K or vitamins of the vitaminB family. Suitable cryoprotectants that may be added to the compositionsof the invention are components that improve the cold tolerance of themicroorganisms, such as mannitol, sorbitol, sodium tripolyphosphate,xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol,trehalose, glucose and fructose. Other additives may include, e.g.,carbohydrates, flavors, minerals, enzymes (e.g. rennet, lactase and/orphospholipase).

In compositions of the invention which comprise Lactobacillus rhamnosusstrain CHCC5366 strain and a Lactobacillus paracasei strain, the ratiobetween the Lactobacillus rhamnosus strain CHCC5366 and theLactobacillus paracasei strain, e.g. the ratio of the concentration ornumber of Lactobacillus rhamnosus bacteria and the concentration ornumber of Lactobacillus paracasei bacteria, preferably amounts from1:100 to 100:1, preferably 1:10 to 10:1.

The antifungal composition of the present invention may be used inconnection with any food, feed and pharmaceutical product which issusceptible to microbial degradation and/or contamination with yeastsand molds. These include, but are not limited to fruits and vegetablesincluding derived products, grain and grain derived products, dairyproducts, meat, poultry, and seafood. In particularly preferredembodiments, the composition is used in connection with dairy productand/or meat and poultry. In a preferred embodiment, the compositions ofthe invention are for use as an additive in the preparation of dairyproducts, such as yoghurt, tvarog, sour cream, cream cheese and thelike.

In a preferred embodiment the compositions of the invention are usedagainst fungi, such as yeasts and molds. This means that thecompositions are used for inhibiting and/or preventing the growth offungi which cause contamination in dairy industry processes, inparticular milk fermentation processes. The compositions of the presentinvention can be used, e.g., for inhibiting and/or preventing the growthof yeasts, such as yeasts of the genera Klyveromyces (e.g., K.marxianus, K. lactis), Pichia (e.g., P. fermentans), Yarrowia (e.g., Y.lipolytica), Candida (e.g., C. sake), and the like; or molds, such asmolds from the genera Penicillium (e.g., P. nalgiovense, P. commune, P.crustosum, P. brevicompactum, P. glabrum), Mucor spp., Cladiosporiumssp., Aspergillus (e.g., A. versicolor), Debaromyces (e.g., D.hansenii), and the like. It is especially preferred to use thecompositions of the invention to inhibit and/or prevent growth of thespecies Klyveromyces marxianus, Yarrowia lipolytica, Penicilliumnalgiovense, Cladiosporium ssp., Penicillium commune, Mucor ssp.,Penicillium brevicompactum, Aspergillus versicolor, Penicilliumcrustosum, Kluyveromyces lactis, and/or Debaromyces hansenii.

The antifungal composition according to the second aspect of the presentinvention may also be used as a pharmaceutical product, preferably onefor treating infections with pathogenic fungi, such as pathogenicyeasts.

In a third aspect, the present invention is directed to a food, feed orpharmaceutical product comprising a Lactobacillus rhamnosus strainaccording to the first aspect of the invention or an antifungalcomposition according to the second aspect of the invention.

In a preferred embodiment the food, feed or pharmaceutical product is afood product.

In a more preferred embodiment such a food product is selected from thegroup consisting of fruits and fruit derived products, vegetable andvegetable derived products, grain and grain derived products, dairyproducts, meat, poultry and seafood and mixtures thereof.

In an even more preferred embodiment the food product is a dairyproduct, preferably a mesophilic or a thermophilic fermented milkproduct, such as fresh cheese, a yoghurt, a sour cream or tvarog.

In another preferred embodiment the food product is meat or poultry.

In a preferred embodiment the food, feed or pharmaceutical product is apharmaceutical product.

Preferably, the pharmaceutical product is a product useful foradministration of the antifungal composition according to a secondaspect of the present invention to a human or an animal to inhibitpathogenic microorganisms and alleviating symptoms related to thepathogenic microorganisms. Examples of such symptoms include symptomsrelated to yeast infection. In such an embodiment, the pharmaceuticalproduct may be a unit dosage form comprising the antifungal composition.Preferably, the unit dosage form is a capsule or a tablet. However, theunit dosage form may also be suitable for application to the mucosa orskin and, thus, be in the form of a paste, cream, ointment and the like.

A fourth aspect of the present invention relates to a method formanufacturing a food, feed or pharmaceutical product according to thethird aspect of the present invention comprising adding at least oneLactobacillus rhamnosus strain according to the first aspect or anantifungal composition according to the second aspect of the inventionduring the manufacture of the food, feed or pharmaceutical product.Preferably, the method also comprises the step of controlling themanufacturing parameters during the manufacturing such that theconcentration of the at least one Lactobacillus rhamnosus strain remainsconstant or is increased.

In a preferred embodiment the concentration of the at least one strainof Lactobacillus rhamnosus is at least 1×10⁶ cfu/g or each at least1×10⁶ cfu/ml of the food, feed or pharmaceutical product, or each atleast 1×10⁵ cfu/cm² of the surface of the food, feed or pharmaceuticalproduct. Preferably, the concentration of the at least one strain ofLactobacillus rhamnosus is at least 5×10⁶ cfu/g or each at least 5×10⁶cfu/ml of the food, feed or pharmaceutical product, or each at least5×10⁵ cfu/cm² of the surface of the food, feed or pharmaceuticalproduct, such as at least 1×10² cfu/g or each at least 1×10² cfu/ml ofthe food, feed or pharmaceutical product, or each at least 1×10⁶ cfu/cm²of the surface of the food, feed or pharmaceutical product, such as atleast 5×10⁷ cfu/g or each at least 5×10⁷ cfu/ml of the food, feed orpharmaceutical product, or each at least 5×10⁶ cfu/cm² of the surface ofthe food, feed or pharmaceutical product.

Where the food, feed or pharmaceutical product is manufactured byaddition of a composition comprising Lactobacillus rhamnosus strainCHCC5366 or a mutant derived thereof and at least one strain ofLactobacillus paracasei, the concentration of Lactobacillus rhamnosusstrain CHCC5366 or the mutant derived thereof and/or the concentrationof the at least one strain of Lactobacillus paracasei is each at least1×10⁶ cfu/g or each at least 1×10⁶ cfu/ml of the food, feed orpharmaceutical product, or each at least 1×10⁵ cfu/cm² of the surface ofthe food, feed or pharmaceutical product. Preferably, the concentrationof Lactobacillus rhamnosus strain CHCC5366 or the mutant derived thereofand/or the concentration of the at least one strain of Lactobacillusparacasei is each at least 5×10⁶ cfu/g or each at least 5×10⁶ cfu/ml ofthe food, feed or pharmaceutical product, or each at least 5×10⁵ cfu/cm²of the surface of the food, feed or pharmaceutical product. In a furtherembodiment, the concentration of Lactobacillus rhamnosus strain CHCC5366or the mutant derived thereof and/or the concentration of the at leastone strain of Lactobacillus paracasei is each at least 1×10⁸ cfu/g oreach at least 1×10⁸ cfu/ml of the food, feed or pharmaceutical product,or each at least 1×10⁷ cfu/cm² of the surface of the food, feed orpharmaceutical product.

In a preferred embodiment, the manufacturing parameters are controlledduring the manufacturing such that the concentration of Lactobacillusrhamnosus strain CHCC5366 (or the mutant derived thereof) and the atleast one strain of Lactobacillus paracasei increases or remainsconstant.

An antifungal composition according to the present invention is mostreadily used by mixing with and/or applying on a blendable food, feed orpharmaceutical product, but should also be effective to treat thesurface of solid food products, or the interior of such products, e.g.by injection. In still other embodiments, the composition may be appliedas a marinade, breading, seasoning rub, glaze, colorant mixture, and thelike, the key criteria being that the antifungal composition beavailable to the surface subject to bacterial degradation andcontamination with yeasts and molds. In still other embodiments, thecomposition may be indirectly placed into contact with the food surfaceby applying the composition to food packaging and thereafter applyingthe packaging to the food surface. The optimum amount to be used willdepend on the composition of the particular food product to be treatedand the method used for applying the composition to the food surface,but can be determined by simple experimentation.

In a much preferred embodiment the method comprises one or morefermentation steps and the at least one Lactobacillus rhamnosus strainor the antifungal composition may be added to the food, feed orpharmaceutical product prior to, during or after such one or morefermentation steps.

Preferably, the method comprises the fermentation of a substrate, suchas a milk substrate, in the presence of at least one Lactobacillusrhamnosus strain according to the invention for a period of timesufficient for the appearance of antifungal activity of the at least oneLactobacillus rhamnosus strain according to the invention. Thisantifungal activity has the effect of inhibiting the development ofyeasts and/or molds in the product fermented with the at least onestrain.

In an even more preferred embodiment the method comprises fermenting amilk substrate with a starter culture comprising at least one strain ofthe genera selected from Lactobacillus, Streptococcus, Lactococcus andLeuconostoc, such as at least one strain of Lactobacillus bulgaricus andat least one strain of Streptococcus thermophilus or such as at leastone strain of Lactococcus lactis subsp. lactis, at least one strain ofLeuconostoc mesenteroides subsp. cremoris and at least one strain ofLactococcus lactis subsp. diacetylactis.

The fifth aspect of the invention relates to a food product obtainableby the method according the fourth aspect of the invention.

The sixth aspect of the present invention is directed to use of aLactobacillus rhamnosus strain according to the first aspect or anantifungal composition according to the second aspect for preparing afood, feed or pharmaceutical product. Preferably, the food productproduced by use of the Lactobacillus paracasei strain according to thefirst aspect or the antifungal composition according to the secondaspect is a cheese, such as a Cottage, Feta, Cheddar, Parmesan,Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheese,brine cheese, Camembert or Brie.

The last aspect of the invention relates to use of a Lactobacillusrhamnosus strain according the first aspect or an antifungal compositionaccording to the second aspect for inhibiting the growth of yeasts andmolds, in particular in food and feed products.

Embodiments of the present invention are described below, by way ofnon-limiting examples.

EXAMPLES Example 1 Challenge Study on Yoghurt with Lactobacillusrhamnosus CHCC5366

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus strain CHCC5366 on the different molds, P. brevicompactum, P.commune, A. versicolor and P. crustosum, yoghurt with 1.5% fat wasprepared:

Homogenized milk (1.5% fat) was heat-treated 95° C.±1° C. for 5 min. in1 L bottles in a water bath and cooled immediately. A commercial starterculture (F-DVS YF-L901 obtainable from Chr. Hansen A/S, Denmark) wasinoculated at 0.02%. The milk was further inoculated with HOLDBAC™ YM-B(20DCU/100 L) or Lactobacillus rhamnosus CHCC5366 (1×10⁷ CFU/ml) and onebottle was used as reference and only inoculated with starter culture.

The milk was fermented at 43° C.±1° C. until pH of 4.60±0.1 was reached.The resulting yoghurt was poured into cups (100 g) and stored at 7°C.±1° C.

One day after preparation of yoghurt different molds were inoculated assurface contaminants in duplicate cups of yoghurt with one spot on thesurface of the yoghurt with a target of 100 spores/spot. Growth of themolds were assessed visually after storage for 45 days at 7° C.±1° C.

The result of the yoghurt test is presented in FIG. 1, showing that P.brevicompactum (M1), P. commune (M6), A. versicolor (M7) and P.crustosum (M10) grow well on yoghurt made from milk fermented only withthe starter culture YF-L901 (top row) or with the starter culture andthe HOLDBAC™ YM-B culture (middle row). In contrast, when Lactobacillusrhamnosus CHCC5366 was present during milk fermentation (bottom row) thegrowth of all tested molds was inhibited.

Example 2 Quantitative Determinations of the Inhibitory Effect ofLactobacillus rhamnosus CHCC5366 Against Debaromyces hansenii

For a quantitative examination of the inhibitory effect of Lactobacillusrhamnosus CHCC5366 on D. hansenii, yoghurt was prepared:

Homogenized milk (1.5% fat) was heat-treated at 95° C.±1° C. for 5 min.in 1 L bottles in a water bath. The milk was cooled immediately. Eachbottle was inoculated with a commercial starter culture (F-DVS YF-L901obtainable from Chr. Hansen A/S, Denmark) at 0.02%. The milk was furtherinoculated with HOLDBAC™ YM-B (20 DCU/100 L) or HOLDBAC™ YM-C (10DCU/100 L) or Lactobacillus rhamnosus CHCC5366 (1×10⁷ CFU/g) and onebottle was used as reference and only inoculated with starter culture.

The milk was fermented at 43° C.±1° C. until pH of 4.60±0.05 wasreached. The resulting yoghurt was poured into cups (approx. 75 ml) andstored at 7° C.±1° C.

The day after preparing the yoghurt, the cups were inoculated induplicate with 0.75 ml/cup of yeast at a target of 20 CFU/g. The yeastwas equally dispersed in the yoghurt. The cups were stored under lid at7° C.±1° C. and analyzed at suitable intervals for the D. hanseniicontamination level by plating 1 ml of yoghurt and further appropriate1-fold dilutions made in saline peptone on Yeast Glucose Chloramphenicol(YGC) agar followed by aerobic incubation for 5 days at 25° C.

As illustrated in FIG. 2, growth of D. hansenii was inhibited in thepresence of Lactobacillus rhamnosus strain CHCC5366 when inoculatedtogether with the starter culture YF-L901 before fermentation. Thestrain caused significantly higher inhibition than the commercialcultures, HOLDBAC™ YM-B and HOLDBAC™ YM-C.

Example 3 Semi-Quantitative Determinations of the Inhibitory Effect ofLb. Paracasei CHCC14676 and Lb. rhamnosus CHCC5366 Alone and inCombination Against Different Mold Contaminants

For the semi-quantitative examinations of Lb. paracasei CHCC14676 andLb. rhamnosus CHCC5366 alone and in combination, an agar-assay was used,resembling the manufacturing process and product of yoghurt:

Homogenized milk (1.5% fat w/v) was heat-treated at 95° C. for fiveminutes and cooled immediately. A commercial starter culture (F-DVSYC-350 obtainable from Chr. Hansen A/S, Denmark) was inoculated at0.02%, and the milk was distributed to 220 ml bottles. The bottles werefurther inoculated with Lb. paracasei CHCC14676, Lb. rhamnosus CHCC5366and a combination of the two strains, respectively, in totalconcentrations of 1×10⁷ CFU/ml. One bottle without further inoculationthan starter culture was used as reference. Furthermore, 5% of apH-indicator of bromcresol purple and bromcresol green was added to allbottles to get an indication of the speed of acidification, and toobtain a blue/green color of the media which would make subsequentgrowth of target yeasts and molds more easily detectable. All bottleswere incubated in a water bath at 43±1° C. and fermented at theseconditions until pH of 4.60±0.1 was reached. After fermentation, thebottles were immediately cooled on ice and vigorously shaken to breakthe coagulum. Then the fermented milk was warmed to a temperature of 40°C. and added to 40 ml of a 5% sterile agar solution that had been meltedand cooled down to 60° C. This solution was then poured into sterilePetri dishes and the plates were dried in a LAF bench for 30 min.

Fully outgrown spore suspensions in appropriate dilutions of theselected molds Penicillium nalgiovese (10×), Penicillium commune (100×),Aspergillus versicolor (100×) and Penicillium crustosum (100×) werespotted on the plates. The plates were incubated at 7° C. and examinedfor the growth of mold at suitable, regular intervals.

Results of the agar-assay are presented in FIG. 3, showing that all ofthe tested molds grew very well on the agar plates from milk fermentedonly with the starter culture (reference). However, when Lb. paracaseiCHCC14676 or Lb. rhamnosus CHCC5366 were present during milkfermentation, the resulting plates strongly reduced growth of all molds.Furthermore, when Lb. paracasei CHCC14676 and Lb. rhamnosus CHCC5366were present in combination during milk fermentation, even strongerinhibition was observed for Penicillium commune, Aspergillus versicolorand Penicillium crustosum spotted on the plates.

Example 4 Challenge study with Lb. rhamnosus CHCC5366 and potassiumsorbate

Pasteurized milk containing 0.17% fat was used to prepare threedifferent batches of yoghurt using the commercial starter culturereferred to in Example 1. The starter culture was inoculated at 0.013%.Prior to fermentation, potassium sorbate (0.023%) was added to the firstbatch, Lactobacillus rhamnosus CHCC5366 (1×10⁷ CFU/g) was added to thesecond batch, while the third batch was used as a reference containingonly the starter culture. Fermentation was carried out at 43° C. until apH of 4.55 was reached. After reaching pH 4.55, the yoghurt was storedat 7° C.

The three different types of yoghurt were distributed into small cupswith approx. 100 g in each and surface inoculated with each of thefollowing molds previously isolated from spoiled fermented milk productsto reach a target inoculation of approx. 1000 spores/cup:

Penicillium solitum,Penicillium palitans,Penicillium discolor,Penicillium spathulatum,Penicillium commune,Penicillium crustosum,Penicillium paneum,Penicillium roqueforti,

The yoghurts were incubated at 7° C. for 60 days, and the outgrowth ofthe different molds was visually monitored by taking pictures at the endof the 60-days period.

The results presented in FIG. 4 demonstrate that the presence ofLactobacillus rhamnosus CHCC5366 dramatically reduced the outgrowth andthe formation of the typical green/blue color of the various molds inthe yoghurt during 60 days storage at 7° C. The inhibitory effect ofLactobacillus rhamnosus CHCC5366 on the extent of outgrowth and colorformation of the molds was found to be considerably stronger than theeffect exerted by the addition of 0.023% potassium sorbate.

Example 5 Challenge Study on Greek Yoghurt with Lactobacillus rhamnosusCHCC5366 and Lactobacillus paracasei CHCC14676 in Combination

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 in combinationon Mucor ssp. Greek yoghurt was prepared substantially as follows:

Pasteurized 1.5% fat milk was inoculated with a commercial starterculture (F-DVS YF-L901 obtainable from Chr. Hansen A/S, Denmark) at0.02%. One batch only inoculated with starter culture was used as areference. Another batch was further inoculated with a combination ofLactobacillus rhamnosus strain CHCC5366 (5×10⁶ CFU/g) and Lactobacillusparacasei strain CHCC14676 (5×10⁶ CFU/g).

The milk was fermented at 43° C.±1° C. to a final pH of 4.55 (6-7hours). Subsequently, the yoghurt was cooled to 25° C.±1° C. with a backpressure of 2 bars and stored at 6° C.±1° C.

One day after preparation of the yoghurt, Mucor ssp. was inoculated assurface contaminant in duplicate cups of yoghurt by applying one spot onthe surface of the yoghurt with a target inoculation concentration of100 spores/spot. The growth of the mold was assessed visually afterstorage for 15 days at 22° C.±1° C.

The results of the Greek yoghurt test are presented in FIG. 5, showingthat Mucor ssp. grows well on the yoghurt made from milk fermented onlywith the starter culture (left cup). In contrast, when Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 were presentduring milk fermentation (right cup) the growth of Mucor ssp. wasinhibited.

Example 6 Second Challenge Study on Greek Yoghurt with a Combination ofLactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676

For the visual examination of the inhibitory effect of the combinationof Lactobacillus rhamnosus CHCC5366 and Lactobacillus paracaseiCHCC14676 on the black bread mold Rhizopus stolonifer the Greek yoghurtwas prepared substantially as follows:

Pasteurized 1.5% fat milk was inoculated with a commercial starterculture (F-DVS YF-L901 obtainable from Chr. Hansen A/S, Denmark) at0.02%. The milk was further inoculated with HOLDBAC™ YM-B (10 DCU/100l), with Lactobacillus rhamnosus strain CHCC5366 (1×10⁷ CFU/g), or witha combination of Lactobacillus rhamnosus strain CHCC5366 (5×10⁶ CFU/g)and Lactobacillus paracasei strain CHCC14676 (5×10⁶ CFU/g). One batchwas used as reference and only inoculated with starter culture.

The milk was fermented at 43° C.±1° C. to a final pH of 4.55 (6-7hours). Subsequently, the yoghurt was cooled to 25° C.±1° C. with a backpressure of 2 bars and stored at 6° C.±1° C.

One day after preparation of the yoghurt, Rhizopus stolonifer wasinoculated as a surface contaminant in duplicate cups of yoghurt byapplying one spot on the surface of the yoghurt with a targetcontamination of 100 spores/spot. Growth of the mold was assessedvisually after storage for 42 days at 7° C.±1° C.

The result of the Greek yoghurt test is presented in FIG. 6, showingthat Rhizopus stolonifer grows well on the yoghurt made from milkfermented only with the starter culture (“R”, i.e. reference) or withthe starter culture and the HOLDBAC™ YM-B culture (YM-B). However, whenLactobacillus rhamnosus CHCC5366 (5366) was present, the growth ofRhizopus stolonifer was inhibited significantly. When Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 were bothpresent during milk fermentation (5366+14676) the growth of Rhizopusstolonifer was inhibited almost completely.

Example 7 Challenge Study on White Brine Cheese with Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 in Combination

For the quantitative examination of the inhibitory effect ofLactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676in combination against Saccharomyces cerevisiae, white brine cheese wasprepared:

Pasteurized and standardised milk was inoculated with a commercialstarter culture (FD-DVS SafeIT-1 obtainable from Chr. Hansen A/S,Denmark) at 40 U per 1000 l milk. The milk was further inoculated withHOLDBAC™ YM-B (10 DCU/100 l) or with a combination of Lactobacillusrhamnosus strain CHCC5366 (5×10⁶ CFU/g) and Lactobacillus paracaseistrain CHCC14676 (5×10⁶ CFU/g). One batch was used as reference and onlyinoculated with starter culture. The milk was rennet-treated at 36°C.±1° C. using Chy-Max plus (obtained from Chr. Hansen A/S, Denmark)with 220 ml per 1000 l for 90 min. before cutting. At pH 6.0 the curdwas pumped to the molds and left for drainage to a final pH of 4.8-4.7(20-24 hours). The cheeses were added into tins with cold brine (8%) andstored at 5° C.±1° C.

One day after preparation of the white brine cheese, the tins wereinoculated in duplicate with yeast at a target of 20 CFU/ml. The yeastwas equally dispersed in the brine. The cups were stored under lid at 7°C.±1° C. for up to 40 days and 10 g were analysed at suitable intervalsfor the Saccharomyces cerevisiae contamination level by platingappropriate 1-fold dilutions made in saline peptone on Yeast GlucoseChloramphenicol (YGC) agar followed by aerobic incubation for 5 days at25° C.

As illustrated in FIG. 7, growth of Saccharomyces cerevisiae wasinhibited in the presence of Lactobacillus rhamnosus CHCC5366 andLactobacillus paracasei CHCC14676 in combination when inoculatedtogether with the starter culture SafeIT-1. The strains causedsignificantly higher inhibition than the commercially available culture,HOLDBAC™ YM-B.

Example 8 Second Challenge Study on White Brine Cheese withLactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676in Combination

For the quantitative examination of the inhibitory effect ofLactobacillus rhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676in combination against Kluyveromyces maxianus, white brine cheese wasprepared as described in example 1, with the exception that the tinswere inoculated with Kluyveromyces maxianus as yeast contaminant.

As illustrated in FIG. 8, growth of Kluyveromyces maxianus was inhibitedin the presence of Lactobacillus rhamnosus CHCC5366 and Lactobacillusparacasei CHCC14676 in combination when inoculated together with thestarter culture SafeIT-1. The strains caused significantly higherinhibition than the commercially available culture HOLDBAC™ YM-B.

Example 9 Third Challenge Study on White Brine Cheese with Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 in Combination

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus CHCC5366 and Lactobacillus paracasei CHCC14676 in combinationagainst P. commune, white brine cheese was prepared as described inexample 1.

7 days after preparation of the white brine cheese, the cheese wasremoved from the brine and P. commune was inoculated as surfacecontaminant on duplicate cheeses with three spots on the surface of thecheese with a target of 100 spores/spot. Growth of two different P.commune isolates were assessed visually after storage for 12 days at 7°C.±1° C. and, subsequently, 16 days at 12° C.±1° C.

The results of the white brine test are presented in FIG. 9, showingthat the two P. commune isolates grow-well on the white brine cheesemade from milk inoculated only with the starter culture (left). Incontrast, when Lactobacillus rhamnosus CHCC5366 and Lactobacillusparacasei CHCC14676 were present during cheese production (right) thegrowth of P. commune was strongly inhibited.

Example 10 Challenge Study on White Brine Cheese with Lactobacillusparacasei CHCC5366

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus CHCC5366 against P. crustosum, white brine cheese wasprepared:

Pasteurized and standardised milk was inoculated with a commercialstarter culture (FD-DVS SafeIT-1 obtainable from Chr. Hansen A/S,Denmark) at 40 U per 1000 l milk. The milk was further inoculated withHOLDBAC™ YM-B (10 DCU/100 l) or Lactobacillus rhamnosus strain CHCC5366(1×10⁷ CFU/g). One batch was used as reference and only inoculated withstarter culture. The milk was rennet-treated at 36° C.±1° C. usingChy-Max plus (obtained from Chr. Hansen A/S, Denmark) with 220 ml per1000 l for 90 min. before cutting. At pH 6.0 the curd was pumped to themolds and left for drainage to a final pH of 4.8-4.7 (20-24 hours). Thecheeses were added into tins with cold brine (8%) and stored at 5° C.±1°C.

7 days after preparation of the white brine cheese, the cheese wasremoved from the brine and P. crustosum was inoculated as surfacecontaminant on duplicate cheeses with three spot on the surface of thecheese with a target of 100 spores/spot. Growth of the P. crustosumisolate was assessed visually after storage for 11 days at 12° C.±1° C.,and, subsequently, 16 days at 12° C.±1° C.

The results of the white brine test are presented in FIG. 10 showingthat the P. crustosum isolate grows-well on the white brine cheese madefrom milk inoculated only with the starter culture (left). In contrast,when Lactobacillus rhamnosus CHCC5366 was present during cheeseproduction (right) the growth of P. crustosum was inhibited.

Example 11 Challenge Study on Yoghurt with Lactobacillus rhamnosusCHCC5366 and CHCC14676 Against Penicillium paneum

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus strain CHCC5366 alone or in combination with Lactobacillusparacasei CHCC14676 on the mold P. paneum, yoghurt with 1.5% fat wasprepared:

Homogenized milk (1.5% fat) was heat-treated 95° C.±1° C. for 5 min. in1 L bottles in a water bath and cooled immediately. A commercial starterculture (F-DVS YF-L901 obtainable from Chr. Hansen A/S, Denmark) wasinoculated at 0.02%. The milk was further inoculated with HOLDBAC™ YM-B(20DCU/100 L), with Lactobacillus rhamnosus CHCC5366 (1×10⁷ CFU/ml)alone, or with a combination of Lactobacillus rhamnosus CHCC5366 (5×10⁶CFU/ml) and Lactobacillus paracasei CHCC14676 (5×10⁶ CFU/ml). One bottlewas used as reference and only inoculated with starter culture.

The milk was fermented at 43° C.±1° C. until pH of 4.60±0.1 was reached.The resulting yoghurt was poured into cups (100 g) and stored at 7°C.±1° C.

One day after preparation of yoghurt different molds were inoculated assurface contaminants in duplicate cups of yoghurt with one spot on thesurface of the yoghurt with a target of 100 spores/spot. Growth of themolds were assessed visually after storage for 28 days at 7° C.±1° C.

The result of the yoghurt test is presented in FIG. 11, showing that P.paneum grows well on yoghurt made from milk fermented only with thestarter culture YF-L901 or with the starter culture and the HOLDBAC™YM-B culture. In contrast, when Lactobacillus rhamnosus CHCC5366 waspresent during milk fermentation the growth of P. paneum was stronglyreduced, and the combination of Lactobacillus rhamnosus CHCC5366 andLactobacillus paracasei CHCC14676 caused even stronger growth inhibitionof P. paneum.

Example 12 Challenge Study on Yoghurt with Lactobacillus rhamnosusCHCC5366 and CHCC14676 at Different Storage Temperatures

For the visual examination of the inhibitory effect of Lactobacillusrhamnosus strain CHCC5366 alone or in combination with Lactobacillusparacasei CHCC14676 on the mold P. crustosum at different storagetemperatures, yoghurt with 1.5% fat was prepared:

Homogenized milk (1.5% fat) was heat-treated 95° C.±1° C. for 5 min. in1 L bottles in a water bath and cooled immediately. A commercial starterculture (F-DVS YF-L901 obtainable from Chr. Hansen A/S, Denmark) wasinoculated at 0.02%. The milk was further inoculated with HOLDBAC™ YM-B(20 DCU/100 L), with Lactobacillus rhamnosus CHCC5366 (1×10⁷ CFU/ml)alone, or with a combination of Lactobacillus rhamnosus CHCC5366 (5×10⁶CFU/ml) and Lactobacillus paracasei CHCC14676 (5×10⁶ CFU/ml). One bottlewas used as reference and only inoculated with starter culture.

The milk was fermented at 43° C.±1° C. until pH of 4.60±0.1 was reached.The resulting yoghurt was poured into cups (100 g) and stored at 7°C.±1° C.

One day after preparation of yoghurt Penicillium crustosum wasinoculated as surface contaminant in duplicate cups of yoghurt with onespot on the surface of the yoghurt with a target of 100 spores/spot.Growth of the molds were assessed visually after storage for 36 days at7° C.±1° C., 12° C.±1° C. or 22° C.±1° C.

The result of the yoghurt test is presented in FIG. 12, showing that P.crustosum grows well on yoghurt made from milk fermented only with thestarter culture YF-L901 or with the starter culture and the HOLDBAC™YM-B culture at all storage temperatures. In contrast, whenLactobacillus rhamnosus CHCC5366 alone or in combination withLactobacillus paracasei CHCC14676 was present during milk fermentation,growth of P. crustosum was completely prevented upon storage both at 7°C.±1° C. and 12° C.±1° C. for 36 days. Upon storage at 22° C.±1° C.growth of P. crustosum was strongly reduced in the presence ofLactobacillus rhamnosus CHCC5366 and even stronger in the presence ofthe combination of Lactobacillus rhamnosus CHCC5366 and Lactobacillusparacasei CHCC14676.

DEPOSITS AND EXPERT SOLUTION

The applicant requests that a sample of the deposited micro-organismsstated below may only be made available to an expert, until the date onwhich the patent is granted.

The Lactobacillus rhamnosus strain CHCC5366 was deposited 2009-10-14 atGerman Collection of Microorganisms and Cell Cultures (Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B,D-38124 Braunschweig and given the accession No.: DSM23035.

The Lactobacillus paracasei strain CHCC12777 was deposited 2011-03-15 atGerman Collection of Microorganisms and Cell Cultures (Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B,D-38124 Braunschweig and given the accession No.: DSM24651.

The Lactobacillus paracasei strain CHCC14676 was deposited 2012-02-02 atGerman Collection of Microorganisms and Cell Cultures (Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B,D-38124 Braunschweig and given the accession No.: DSM25612.

The deposit was made according to the Budapest treaty on theinternational recognition of the deposit of microorganisms for thepurposes of patent procedure.

REFERENCES

-   EP0221499-   EP0576780-   EP1442113-   U.S. Pat. No. 5,378,458-   EP11161609.0-   Kosikowski, F. V. and Mistry, V. V., “Cheese and Fermented Milk    Foods”, 1997, 3rd Ed. F. V. Kosikowski, L.L.C. Westport, Conn.

1. A Lactobacillus rhamnosus strain selected from the group consistingof the Lactobacillus rhamnosus strain CHCC5366 that was deposited withthe German Collection of Microorganisms and Cell Cultures (DSMZ) underaccession No. DSM23035 and mutant strains derived thereof, wherein themutant strains have substantially the same or improved antifungalproperties as the strain deposited under accession No. DSM23035.
 2. Anantifungal composition comprising at least one Lactobacillus rhamnosusstrain according to claim
 1. 3. The antifungal composition according toclaim 2 further comprising at least one strain of Lactobacillusparacasei.
 4. The antifungal composition according to claim 3, whereinthe at least one strain of Lactobacillus paracasei is selected from thegroup consisting of Lactobacillus paracasei CHCC12777 with accession no.DSM24651 and Lactobacillus paracasei CHCC14676 with accession no.DSM25612 and mutant strains derived thereof, wherein the mutant strainshave substantially the same or improved antifungal properties as one ofthe strains deposited under accession No. DSM24651 or DSM25612.
 5. Afood, feed or pharmaceutical product comprising a Lactobacillusrhamnosus strain according to claim
 1. 6. The food product according toclaim 5 comprising an amount of the Lactobacillus rhamnosus straineffective for imparting antifungal properties to the food product. 7.The food product according to claim 5, wherein the food product isselected from the group consisting of fruits and fruit derived products,vegetables and vegetable derived products, grain and grain derivedproducts, dairy products, meat, poultry and seafood and mixturesthereof.
 8. The food product according to claim 7, wherein the foodproduct is a dairy product selected from the group consisting ofmesophilic and thermophilic fermented milk products.
 9. A method formanufacturing a food, feed or pharmaceutical product comprising addingat least one Lactobacillus rhamnosus strain according to claim 1 to thefood, feed or pharmaceutical product during manufacture and controllingthe manufacturing parameters during the manufacturing such that theconcentration of the at least one Lactobacillus rhamnosus strain remainsconstant or is increased.
 10. The method according to claim 9, whereinthe concentration of the at least one Lactobacillus rhamnosus strain isat least 1×10⁶ cfu/g or each at least 1×10⁶ cfu/ml of the food, feed orpharmaceutical product, or at least 1×10⁵ cfu/cm² of the surface of thefood, feed or pharmaceutical product.
 11. The method according to claim9, comprising (a) adding an antifungal composition comprising at leastone Lactobacillus rhamnosus strain according to claim 1 and at least onestrain of Lactobacillus paracasei during the manufacture of the food,feed or pharmaceutical product such that the concentration of the atleast one strain of Lactobacillus rhamnosus and/or the at least onestrain of Lactobacillus paracasei is each at least 1×10⁶ cfu/g or atleast 1×10⁶ cfu/ml of the food, feed or pharmaceutical product, or atleast 1×10⁵ cfu/cm² of the surface of the food, feed or pharmaceuticalproduct, and (b) controlling the manufacturing parameters during themanufacturing such that the concentration of the at least one strain ofLactobacillus rhamnosus and/or the at least one strain of Lactobacillusparacasei increases or remains constant.
 12. The method according toclaim 9, wherein the method comprises one or more fermentation steps.13. The method according to claim 12, wherein the method comprisesfermenting a milk substrate with a starter culture comprising at leastone strain of the genera selected from Lactobacillus, Streptococcus,Lactococcus and Leuconostoc.
 14. A food product obtained by the methodaccording to claim
 9. 15. A method for preparing a food, feed orpharmaceutical product comprising adding a Lactobacillus rhamnosusstrain according to claim 1 to the food, feed or pharmaceutical productduring manufacture.
 16. A method for inhibiting the growth of yeasts andmolds in food or feed products comprising adding a Lactobacillusrhamnosus strain according to claim 1 to the food or feed product.
 17. Amethod for preparing a food, feed or pharmaceutical product comprisingadding an antifungal composition according to claim 2 to the food, feedor pharmaceutical product during manufacture.
 18. A method forinhibiting the growth of yeasts and molds in food or feed productscomprising adding an antifungal composition according to claim 2 to thefood or feed product.